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5 Temperature Minimum Temperature: Temperature below which growth ceases, or lowest temperature at which microbes will grow. Optimum Temperature: Temperature at which its growth rate is the fastest. Maximum Temperature: Temperature above which growth ceases, or highest temperature at which microbes will grow.

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11 pH and Microbial Growth The acidity or alkalinity of an environment can greatly affect microbial growth. Most organisms grow best between pH 6 and 8, but some organisms have evolved to grow best at low or high pH. The internal pH of a cell must stay relatively close to neutral even though the external pH is highly acidic or basic. –Acidophiles : organisms that grow best at low pH ( Helicobacter pylori, Thiobacillus thiooxidans ) –Alkaliphiles : organismsa that grow best at high pH ( Vibrio cholera) –Most of pathogenic bacteria are neutrophiles

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13 Osmotic Effects on Microbial Growth Osmotic pressure depends on the surrounding solute concentration and water availability Water availability is generally expressed in physical terms such as water activity (a w ) Water activity is the ratio of the vapor pressure of the air in equilibrium with a substance or solution to the vapor pressure of pure water ( aw 1.00). a w = P solu P water

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14 Environmental factors and growth 1. Osmotic Effect and water activity organisms which thrive in high solute – osmophiles organisms which tolerate high solute – osmotolerant organisms which thrive in high salt – halophiles organisms which tolerate high salt – halotolerant organisms which thrive in high pressure – barophiles organisms which tolerate high pressure – barotolerant

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16 Halophiles and Related Organisms In nature, osmotic effects are of interest mainly in habitats with high salt environments that have reduced water availability Halophiles : have evolved to grow best at reduced water potential, and some (extreme halophiles e.g. Halobacterium, Dunaliella ) even require high levels of salts for growth. Halotolerant : can tolerate some reduction in the water activity of their environment but generally grow best in the absence of the added solute Xerophiles : are able to grow in very dry environments

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18 Microbial Nutrition Why is nutrition important? –The hundreds of chemical compounds present inside a living cell are formed from nutrients. Macronutrients : elements required in fairly large amounts Micronutrients : metals and organic compounds needed in very small amounts

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19 Main Macronutrients Carbon (C, 50% of dry weight) and nitrogen (N, 12% of dry weight) Autotrophs are able to build all of their cellular organic molecules from carbon dioxide Nitrogen mainly incorporated in proteins, nucleic acids Most Bacteria can use Ammonia -NH 3 and many can also use NO 3 - Nitrogen fixers can utilize atmospheric nitrogen (N 2 )

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22 Classification of organisms based on sources of C and energy used

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23 Nitrogen requirements Although many biological components within living organisms contain N, and N 2 is the most abundant component of air, very few organisms can “fix” or utilize N 2 by converting it to NH 3 N is often growth limiting as organisms must find source as NH 4 + for biosynthesis Photosynthetic organisms and many microbes can reduce NO 3 - to NH 4 +

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45 Chemical Composition of Culture Media 1.Synthetic Media Chemically defined Contain pure organic and inorganic compounds Exact formula (little variation) 2.Complex or Non-synthetic Media Contains at least one ingredient that is not chemically definable (extracts from plants and animals) No exact formula / tend to be general and grow a wide variety of organisms

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46 Selective Media Contains one or more agents that inhibit the growth of a certain microbe and thereby encourages, or selects, a specific microbe. Example: Mannitol Salt Agar [MSA] encourages the growth of S. aureus. MSA contain 7.5% NaCl which inhibit the growth of other Gram +ve bacteria

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47 Growth of Staphylococcus aureus on Mannitol Salt Agar results in a color change in the media from pink to yellow.

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48 Differential Media Differential shows up as visible changes or variations in colony size or color, in media color changes, or in the formation of gas bubbles and precipitates. Example: Spirit Blue Agar to detect the digestion of fats by lipase enzyme. Positive digestion (hydrolysis) is indicated by the dark blue color that develops in the colonies. Blood agar for hemolysis (α,β,and γ hemolysis), EMB, MacConkey Agar, …etc.

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49 Growth of Staphylococcus aureus on Manitol Salt Agar results in a color change in the media from pink to yellow.

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53 Growth of Staphylococcus aureus on Manitol Salt Agar results in a color change in the media from pink to yellow.

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54 Laboratory Culture of Microorganisms Microorganisms can be grown in the laboratory in culture media containing the nutrients they require. Successful cultivation and maintenance of pure cultures of microorganisms can be done only if aseptic technique is practiced to prevent contamination by other microorganisms.

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55 Microbial growth Microbes grow via binary fission, resulting in exponential increases in numbers The number of cell arising from a single cell is 2 n after n generations Generation time is the time it takes for a single cell to grow and divide

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56 Binary Fission

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57 Rapid Growth of Bacterial Population

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58 Growth curve During lag phase, cells are recovering from a period of no growth and are making macromolecules in preparation for growth During log phase cultures are growing maximally Stationary phase occurs when nutrients are depleted and wastes accumulate (Growth rate = death rate) During death phase death rate is greater than growth rate

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60 Viable counts Each colony on plate or filter arises from single live cell Only counting live cells

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61 Direct Count Pour Plate

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63 Direct Count Spread or Streak Plate

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65 Microscopic counts Need a microscope, special slides, high power objective lens Typically only counting total microbe numbers, but differential counts can also be done

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66 Turbitity Cells act like large particles that scatter visible light A spectrophotometer sends a beam of visible light through a culture and measures how much light is scattered Scales read in either absorbance or % transmission Measures both live and dead cells

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67 Inoculation Sample is placed on sterile medium providing microbes with the appropriate nutrients to sustain growth. Selection of the proper medium and sterility of all tools and media is important. Some microbes may require a live organism or living tissue as the inoculation medium.

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68 Incubation An incubator can be used to adjust the proper growth conditions of a sample. Need to adjust for optimum temperature and gas content. Incubation produces a culture – the visible growth of the microbe on or in the media

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69 Isolation The end result of inoculation and incubation is isolation. On solid media we may see separate colonies, and in broth growth may be indicated by turbidity. Sub-culturing for further isolation may be required.